Functional foods comprising diamine oxidase and uses thereof

ABSTRACT

The present invention relates to functional foods comprising DAO and their use for the prevention or treatment of diseases or pathological conditions associated with high levels of histamine in blood, in particular for the prevention or treatment of migraine, chronic fatigue, fibromyalgia, spondylitis and pain caused by muscle contractures.

FIELD OF THE INVENTION

The present invention relates to functional foods comprising diamineoxidase (DAO) for the treatment or prevention of diseases orpathological conditions associated with high histamine levels in blood,in particular for the treatment or prevention of migraine, chronicfatigue, fibromyalgia, spondylitis and pain caused by musclecontractures.

BACKGROUND OF THE INVENTION

Histamine [2(4-imidazolyl)-ethylamine] is an important mediator in manybiological processes including inflammation, gastric acid secretion,neuromodulation and immune function regulation. Because of its potentpharmacological activity, even at very low concentrations, it isnecessary to carefully regulate the synthesis, transport, storage,release and degradation of histamine to prevent undesirable reactions.High concentrations of free histamine in the circulation triggerunwanted effects such as headaches, blocked-up nose or rhinorrhoea,respiratory tract obstructions, tachycardia, gastric and intestinaldisorders, swelling of eyelids, skin rashes, low blood pressure,bronchospasm, etc.

Histamine is produced by the human body and stored in an inactive formin the metachromatic granules of mast cells and basophil leukocytes,where it is available for immediate release. The highest concentrationsof histamine are measured in the lung. After release, histamine becomesan extraordinarily potent mediator of a number of physiological andpathophysiological processes, often also through interaction withcytokines.

Histamine can also enter the body from the outside, since it isgenerated by microbiological action in the course of the processing offood and therefore is present in substantial quantities in manyfood-types and fermented beverages such as cheese, wine, canned fish andsauerkraut.

Histamine is formed by decarboxylation of the L-histidine amino acid ina reaction catalyzed by the histidine decarboxylase enzyme (HDC). Themain routes of histamine inactivation in mammals are methylation of theimidazole ring, catalyzed by histamine-N-methyltransferase (NMT) toobtain N-methylhistamine, and the oxidative deamination of the primaryamino group, catalyzed by diamine oxidase (DAO) to obtain imidazoleacetaldehyde.

Both ways of degradation are essential to the organism: DAO eliminateshistamine, which, for example, has been absorbed through the food in theintestinal tract and NMT controls the transmission of histaminergicsignals in the nervous system.

The main function of DAO is to prevent histamine ingested through foodfrom reaching the bloodstream from the intestine.

In addition to histamine, DAO can degrade other biogenic amines, such asputrescine, spermidine and cadaverine. It has a molecular weight ofapproximately 182 kDa and a carbohydrate ratio of 11%. It belongs to thetype of copper-containing amine oxidases which catalyze the oxidativedeamination of primary amines to obtain aldehydes, ammonia and hydrogenperoxide. DAO uses molecular oxygen to oxidatively deaminate histamineto imidazole acetaldehyde, ammonia and hydrogen peroxide.

DAO is found mainly in the small intestine, liver, kidneys and bloodleukocytes. Pregnant women have a DAO level in blood about 500 to 1000times higher than non-pregnant women, because DAO is additionally formedin the placenta. Histamine is produced continuously in humans and isexcreted via the intestine, being degraded as it passes through theintestinal mucosa by DAO existing there.

DAO is a sensitive enzyme which can be inhibited by various substances,such as other biogenic amines, alcohol and its acetaldehyde degradationproduct, as well as by various medications.

DAO deficiency has been associated as a possible migraine triggerfactor, a condition prevailing in women. An excess of histamine in thebody due to lack of DAO, increases the risk of migraine.

EP 132674 discloses a process for the enzymatic separation of freeamines of food with a high content thereof, such as chocolate,cheese—especially matured cheese—, salami, wine and yeast extracts usingamino oxidase enzymes, in particular DAO, obtained from Aspergillusniger organisms in the presence of molecular oxygen. The presence ofthese free amines in certain foods is considered to cause migraines.

U.S. Pat. No. 4,725,540 discloses a process for the preparation of DAOfrom a DAO-producing microorganism such as Candida crusei or lacticacid-producing bacteria in a nutrient medium, so that the produced DAOis capable of degrading the histamine to a pH between neutral and about4.

The availability of new foods with health benefits is introduced in the60s. Since then new types of food designed for gluten-free, low-sodium,low-calorie diets, etc. have emerged on the market. This food is alsoknown as “Functional food”, including any food in a natural or processedform which in addition to its nutritional components contains additionalcomponents that promote a person's health, physical ability and mentalcondition. Its description as functional relates to the bromatologicalconcept of “functional property”, i.e. the characteristic of a certainfood that has shown to have a beneficial effect on one or more specificbody functions, without reference to its nutritional value. Thus, it isrelevant to health and welfare and helps to reduce the risk of a givendisease.

The United States is one of the countries where functional foods aremore solidly established intended for the prevention of diseases in thepopulation, such as cereal bars intended for middle-aged womensupplemented with calcium to prevent osteoporosis, or protein soya toreduce the risk of breast cancer, or folic acid for a healthier heart,or energy biscuits containing protein, zinc and antioxidants.

In Europe various functional foods with different therapeutic purposesare also known, such as:

-   -   Eggs enriched with omega-3 essential fatty acids, which help        reduce the risk of heart disease.    -   Milk and yogurt fermented with probiotic cultures to help        digestion.    -   Cereals with folic acid to help reduce the number of children        born with spina bifida.    -   Margarines with phytosterols that reduce cholesterol and        decrease the risk of heart disease.

Patent application EP 865737 discloses dairy-based foods enriched with abalanced mixture of soluble and insoluble calcium salts.

Patent application WO 9631130 discloses beverage concentrates whichcomprise a source of calcium and vitamin D.

Patent application WO 9207475 discloses a salad dressing product in theform of an oil emulsion containing a source of calcium.

U.S. Pat. No. 4,851,243 discloses calcium-enriched dairy products in theform of a stable suspension.

However, functional foods comprising DAO to help prevent or treatdiseases and conditions associated with high histamine levels in bloodare not known.

DEFINITIONS

“Functional food” (in the literature often abbreviated as FF) is foodprepared not only for its nutritional properties but also to fulfil aspecific function such as to improve health and reduce the risk ofdisease. To this end, biologically active components are added such asminerals, vitamins, fatty acids, dietary fibre and antioxidants, etc.This operation of adding exogenous nutrients is also calledfortification. This type of food is an emerging field of food sciencewith ample possibilities in food research. Some of the achievementslisted in the scientific literature and marketing of food products arethe improvement of gastrointestinal functions, the contribution of redoxand antioxidant systems, as well as the modification of themacronutrient metabolism. This type of food covers a wide spectrum ofpossibilities that can range from just cereals and their derivatives toall kinds of dairy products.

“Non-plant origin” refers to all DAO not derived from plants but fromanimal organisms or other non-plant organisms. Thus, this definitioncovers all isolated DAO from living organisms.

“Plant origin” means all DAO obtained from plant organisms.

“Biotechnological origin” means all DAO recombinantly prepared in cellculture or in non-plant organisms of any type isolating the DNA for DAO.

In the present invention, “prevention” means preventing the onset of adisease or pathological condition associated with a high level ofhistamine in blood and characterized by pain increase, includingmigraine, chronic fatigue, fibromyalgia, spondylitis and musclecontractures.

In the present invention, “treatment” means clinical intervention in anattempt to alter the natural evolution of the individual and isperformed during the course of clinical pathology. The desired effectsof treatment include prevention of disease recurrence, symptom relief,reduction of any direct or indirect pathological consequence of thedisease, decreased rate of progression of the disease, improvement orpartial cure of the pathological condition and remission or improvedprognosis.

SUMMARY OF THE INVENTION

The problem to be solved by the present invention is the absence offunctional foods comprising DAO which would allow prevention ortreatment of diseases or pathological conditions associated with highhistamine levels in blood. In particular these diseases are migraine,chronic fatigue, fibromyalgia, spondylitis and pain caused by musclecontractures.

This food can be any type of dairy product. The dairy product may beyogurt, milky beverages, a confectionery product, ice-cream or any otherbeverage such as juice for instance. It can also be any dairy substitutesuch as soya, oat, almond or rice milk, etc.

This food can also be any type of cereal, muesli or the like. DAO canalso be added to vegetable oils, animal and vegetable fats, butter,margarine, etc.

DAO can be added to foods in free form, in the form of powder,lyophilized powder, microgranules, microcapsules, nanocapsules orliposomes.

DAO in the aforementioned forms can also be added to nutritionalsupplements such as capsules comprising omega-3 fatty acids, vitamins,minerals, amino acids, etc.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a functional food comprising DAO.

One embodiment of the present invention is a dairy-type functional foodcomprising DAO in free form, in the form of powder, lyophilized powder,microgranules, microcapsules, nanocapsules or liposomes.

Another embodiment of functional food is dairy substitutes such as soya,oat, almond or rice milk, etc. comprising DAO in free form, in the formof powder, lyophilized powder, microgranules, microcapsules,nanocapsules or liposomes.

Functional foods comprising cereal and the like comprising DAO in freeform, in the form of powder, lyophilized powder, microgranules,microcapsules, nanocapsules or liposomes also constitute anotherembodiment of the present invention.

Incorporating DAO in free form, in the form of powder, lyophilizedpowder, microgranules, microcapsules, nanocapsules or liposomes intofood and vitamin supplements is also considered as another object of thepresent invention.

DAO concentrations in these foods are between 0.01 and 5% by weight ofDAO as regards to the total weight of the food, preferably between 0.5and 1%.

DAO used in the present invention may be either of a biotechnologicalorigin or a plant or animal extraction.

DAO used in the present invention may have a gastrointestinal protectionto prevent its degradation in the stomach.

The enteric coating layer covering the microgranules disintegrates ordissolves rapidly in a neutral or alkaline medium.

Microgranule cores can be inert with a sugar or similar base onto whichDAO is applied or said cores may already contain DAO mixed with otherexcipients. Said excipients may be binders, surfactants, fillers,disintegrants, alkaline additives or other pharmaceutically acceptableingredients alone or in a mixture. The binders may be cellulose typesuch as hydroxypropylmethylcellulose, hydroxypropylcellulose andcarboxymethylcellulose sodium, polyvinylpyrrolidone, sugars, starchesand other pharmaceutically acceptable substances based on their cohesiveproperties. Suitable surfactants are found in the groups of acceptablenon-ionic or ionic surfactants, such as for example sodium laurylsulphate.

Alternatively, DAO can be mixed with alkaline compounds and be furthermixed with suitable constituents to be formulated into a core material.Said core materials may be produced by extrusion/spheronization orcompression using different process equipment.

DAO can also be mixed with pharmaceutically acceptable alkali such asphosphoric acid salts and sodium, potassium, calcium, magnesium andaluminium, carbonic acid, citric acid or other suitably weak organic orinorganic acids; a coprecipitate of aluminium hydroxide/sodiumbicarbonate; substances normally used in antiacid preparations such asaluminium, calcium and magnesium hydroxide, magnesium oxide or compositesubstances, such as Al₂O₃.6MgO.CO₂.12H₂O, (Mg₆Al₂ (OH)₁₆CO3.4H₂O,MgO.Al₂O₃.2SiO₂.nH₂O or similar compounds; pH buffering substances suchas trihydroxymethylaminomethane, basic amino acids and their salts orother pharmaceutically acceptable pH buffer substances.

The enteric coating layers may contain pharmaceutically acceptableplasticizers to obtain the desired mechanical properties of flexibilityand hardness. These plasticizers may be, for example, triacetin, citricacid esters, phthalic acid esters, cetyl alcohol, polyethylene glycols,polysorbates or other plasticizers.

The microgranules are added to solid functional foods just by mixing andto liquid foods by shaking them together so they remain in suspension.

The present invention also relates to the use of functional foodcomprising DAO of this invention for the prevention or treatment ofdiseases or pathological conditions associated with high histaminelevels in blood.

The present invention also relates to the use of the functional foodcomprising DAO of this invention for the prevention or treatment ofmigraine.

The present invention also relates to the use of functional foodcomprising DAO of this invention for the prevention or treatment ofchronic fatigue, fibromyalgia, spondylitis and pain from musclecontractures

Furthermore, the present invention also relates to a functional foodcomprising DAO for use in the prevention or treatment of diseases orpathological conditions associated with high histamine levels in blood.The present invention also relates to a functional food comprising DAOfor use in the prevention or treatment of migraine. The presentinvention also relates to a functional food comprising DAO for theprevention or treatment of chronic fatigue, fibromyalgia, spondylitisand pain caused by muscle contractures.

The present invention also relates to the use of DAO for the manufactureof a functional food according to any of the embodiments of the presentinvention for the prevention or treatment of diseases or pathologicalconditions associated with high histamine levels in blood. In particularthe disease or pathological condition is migraine, chronic fatigue,fibromyalgia, spondylitis and pain caused by muscle contractures.

EXAMPLES

The following examples further illustrate the present invention.

Example 1

Microgranules are prepared containing 4% of DAO, according to thefollowing formula

DAO  4 mg Manitol 40 mg Microcrystalline cellulose 25 mg Hydroxypropylcellulose 10 mg Corn starch 10 mg Citric acid  6 mg

The microgranules are coated with hydroxypropyl methylcellulose.

Example 2

The following skimmed milk suspension containing DAO is prepared:

-   -   A. 10 g of microgranules containing 10% of DAO    -   B. A dispersion of 2.5 g of gum arabic in 100 g of water and        thoroughly hydrated    -   C. 830 g of skimmed milk prepared with skimmed milk powder    -   D. 0.15 g of kappa carrageenan

The pH of the final product is adjusted to 6.6 to 7.0, UHT sterilizedand homogenised. The milk was stable showing no sedimentation duringstorage from 2° C. up to room temperature.

Example 3

The following almond milk suspension containing DAO is prepared:

-   -   A. 15 g of microgranules containing 10% of DAO    -   B. A dispersion of 2.5 g of gum arabic in 100 g of water and        thoroughly hydrated    -   C. 830 g of almond milk made from almond milk powder    -   D. 0.15 g of kappa carrageenan

The pH of the final product is adjusted to 6.6 to 7.0, UHT sterilizedand homogenised. The milk was stable showing no sedimentation duringstorage from 2° C. up to room temperature.

Example 4

A food supplement is prepared in capsule form incorporating the DAOmicrogranules of Example 1. For a 350-mg capsule:

-   -   A. 25 mg of microgranules containing 10% of DAO    -   B. 125 mg of fish oil powder containing 20% of omega-3 fatty        acid    -   C. 1.25 mg of vitamin PP (niacin)    -   D. 5 mg of magnesium stearate    -   E. 93.75 mg of Emcocel®

Example 5

A vegetable beverage containing DAO microgranules is prepared with thefollowing components:

-   -   A. 1.5 g of microgranules containing 10% of DAO    -   B. 1 g of stabilizer (mixture of pectin, guar and carob seed        flour 1:1:1)    -   C. 70 g of fibrous substance from vegetables (mainly tomatoes)    -   D. 100 g of mashed vegetables made with tomatoes and carrots    -   E. 50 g of vegetable juice concentrate (tomato, pepper, celery        and beetroot)    -   F. 90 g of fructose syrup    -   G. 20 g of lactic-fermented concentrate    -   H. 2 g of natural vegetable and spice flavouring

All components are completed to 600 g with water and mixed. Thenhomogenized with stirring, degassed and filled into bottles.

Example 7

The functional foods comprising DAO, which are an object of the presentinvention, are administered to a total of 48 subjects (20 men and 28women, aged between 23 and 65), the administering protocol being oncedaily for 4 weeks. These patients were additionally given a diet rich inhigh-histamine-content foods (cheeses, chocolates, canned fish, etc). Inparallel histamine levels in blood were measured in a group of 30subjects who were given the same diet yet based on food without any DAOcontent.

To verify the influence of food comprising DAO in the histamine levelsin blood, analyses on histamine levels before and after a 4-week periodof food intake were conducted. Values of histamine in blood wereconsidered normal when between 2 and 20 micrograms of histamine perdecilitre of blood. Values of histamine in blood were considered highwhen greater than 20 micrograms of histamine per decilitre of blood.

Subjects not Subjects given given food with food with DAO: 48 DAO: 30Histamine level in 2-20 >>20 blood micrograms/0.1 L micrograms/0.1 L

1. A functional food comprising DAO.
 2. A functional food comprisingDAO, according to claim 1, wherein the DAO can be added to the food infree form, in the form of powder, lyophilized powder, microcapsules,nanocapsules or liposomes.
 3. A functional food according to claim 1,wherein the DAO is both of biotechnological origin and from plant oranimal extraction.
 4. A functional food according to claim 1, whereinthe DAO includes gastrointestinal protection.
 5. A functional foodaccording to claim 1, wherein the food is a dairy-based product such asyogurt, milky beverages, a confectionery product, ice-cream or anotherbeverage such as, for example, juice; a non-dairy-based product such assoya, oat, almond or rice milk; cereals and the like.
 6. A functionalfood according to claim 1, wherein the food is a nutritional or vitaminsupplement.
 7. A functional food according to claim 1, wherein the DAOis present in a percentage comprised between 0.01 and 5% by weight ofDAO as regards to the total weight of the food.
 8. A method ofpreventing or treating a disease or pathological condition associatedwith high histamine levels in blood, comprising administering afunctional food comprising DAO.
 9. A method of preventing or treatingmigraine, comprising administering a functional food comprising DAO. 10.A method of preventing or treating chronic fatigue, fibromyalgia,spondylitis and pain caused by muscular contractures, the methodcomprising administering a functional food comprising DAO.
 11. Themethod according to claim 8, wherein the DAO is present in a percentagecomprised between 0.01% and 5% by weight of DAO as regards to the totalweight of the food.
 12. The method according to claim 9, wherein the DAOis present in a percentage comprised between 0.01% and 5% by weight ofDAO as regards to the total weight of the food.
 13. The method accordingto claim 10, wherein the DAO is present in a percentage comprisedbetween 0.01% and 5% by weight of DAO as regards to the total weight ofthe food.